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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
  • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2820/00—Vectors comprising a special origin of replication system
  • C12N2820/002—Vectors comprising a special origin of replication system inducible or controllable
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2820/00—Vectors comprising a special origin of replication system
  • C12N2820/60—Vectors comprising a special origin of replication system from viruses
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
  • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
  • C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible

Definitions

• the present invention is directed to a conditional gene vector system and to a host cell, which has been transfected with such a vector system.
• the present invention is further directed to a combined preparation comprising the vector system of the invention and an interfering agent.
• a pharmaceutical composition and its use in the treatment of hemophilia, diabetes, rheumatoid arthritis, genetic immunodeficiency, and graft versus host disease is provided.
• Recombinant nucleic acids are the basis of gene vectors, which are employed for gene and immune therapy of various human diseases. Most gene vectors encompass viral or metazoan genetic components, which provide the necessary c/s-acting elements such as promoters and enhancers to express one or more genes of therapeutic interest. In addition, certain gene vectors which are designed on virus blueprints also carry the essential regulatory signals involved in packaging of nucleic acids into viral structural components as well as replicative elements for the amplification of the viral vector genomes.
• gene vectors can be transiently present, only, or maintained for a long period of time.
• the genetic information is either rapidly lost through spontaneous degradation by cellular nucleases or maintained by integrating the genetic information into the chromosome of the recipient cell.
• gene vectors are commonly employed, which promote chromosomal integration of their genetic information.
• all retroviruses and adeno-associated viruses integrate as proviruses during this part of the viral life cycles to establish a latent or persistent state.
• DNA- based gene vectors can also integrate chromosomally. Integration of foreign DNA disrupts the genetic integrity of the host chromosome.
• All gene vectors which are characterized by this feature, are derived from recombinant DNA plasmids, which carry autonomous replicons (Piechaczek et al., 1999; Schaarschmidt et al., 2004; Sugden and Leight, 2001). Replicons are genetic units, which mediate DNA replication of the gene vector DNAs often in synchrony with the replication of the host cell chromosome.
• Epstein-Barr virus is the latent origin latent origin of DNA replication of this human herpesvirus. Plasmids containing this origin of DNA replication replicate akin to the genome of its host cell and are maintained extrachromosomally. Consequently, this plasmid replicon has been exploited as recombinant gene vectors (Sugden and Leight, 2001 ).
• the EBV plasmid replicon termed oriP, supports efficient DNA replication in cells selected to retain it at several copies when the viral gene product EBNAl is provided. Recombinant plasmids containing oriP are replicated once per cell cycle during S phase and are efficiently portioned to daughter cells. Only two components, oriP in cis and EBNAl in trans, are required the cell contributes everything else.
• the replicon oriP consists of two essential elements, the family of repeats (FR) and a structure called dyad symmetry element (DS, Fig. 1). Initiation of DNA replication takes place at or near DS to which components of the cellular pre-replication complex (pre-RC) including ORC and MCM proteins are recruited (Ritzi et al., 2003; Schepers et al., 2001). It is likely that EBNAl, which binds to four low affinity binding sites within DS, contributes to the recruitment of pre-RC, perhaps in conjunction with other structural features of DS.
• the FR element is an array of 20 high affinity binding motifs for EBNAl and is dedicated to function in nuclear retention of oriP.
• EBNAl is a viral protein, which interacts with oriP directly and is characterized by a modular design.
• the carboxy-terminal half encompasses aa residues 459 to 604 of the prototype EBNAl gene product of the B95.8 strain of EBV (Baer et al., 1984), which constitutes a dimerization and DNA-binding domain (Fig. 2).
• the amino-terminal half of EBNAl in particular amino acid residues 1 to 89 and 322 to 379 mediate association with cell chromosomes (Marechal et al., 1999) and is essential for plasmid maintenance and transcriptional activation (Yates and Camiolo, 1988).
• the remaining domains are a glycin- alanin repeat unit (aa 90 to 327) supposedly involved in protein degradation, a nuclear localization signal (aa 379 to 386), and an acidic activation domain (aa 605 to 641)(Kieff and Rickinson, 2001 for a review).
• chromosome association of EBNAl triggered an approach in which its amino-terminal half was replaced with cellular proteins known to confer chromatin binding and association to mitotic chromosomes (Hung et al., 2001).
• chimeric gene products consisting of the cellular histone Hl or HMGAIa proteins fused to amino acid residues 379 to 641 of EBNAl functionally replaced the wild-type (wt) EBNAl allele with respect to both plasmid replication and maintenance (Sears et al., 2003).
• the solution of the present invention is the first approach to establish extrachromosomal gene vector plasmids, whose maintenance in a specific target cell can be controlled in cis by, e.g., a small molecular weight compound.
• AT-hook domains are a hallmark of HMG family members (Harrer et al., 2004) of which HMGAI a binds to interphase and mitotic chromatin throughout the cell cycle. HMGAI a was also recognized to promote stable maintenance of oriP wt plasmids when fused to the DNA binding domain of EBNAl (Hung et al., 2001).
• This invention further shows that synthetic, o ⁇ ' P-like plasmid replicons can be engineered such that viral factors or domains become entirely dispensable.
• the four low affinity binding sites within DS (Fig. 1) can be replaced with tetO sites such that fusion proteins harboring the TetR DNA binding domain can recruit the pre-replicative complex to the origin of DNA replication as well.
• oriP ⁇ ike plasmid replicons can be maintained extrachromosomally for weeks.
• the inventors also have evidence that doxycycline leads to a precipitous loss of such plasmids presumably because it abrogates plasmid maintenance as well as DNA replication.
• Such on/Mike plasmid replicons rely on fusion proteins with the TetR DNA binding domain, only, and do not require EBNAl for their DNA replication.
• gene vector plasmids can be designed which carry the oriP-l ⁇ ke replicon, an expression cassette encoding TetR:TetR:HMGAla, and one or more additional genes of interest (Fig. 7 for an example).
• Tetracycline-regulated gene expression has been demonstrated in vivo (Schonig et al., 2002) indicating that gene vectors, which can be regulated in cis are likely to be functional in vivo as well. Mutants of the tetR gene with an inverse phenotype bind to tetO motifs exclusively in the presence of the drug (Gossen et al., 1995; Urlinger et al., 2000) and are expected to be functional in the present system, too.
• TetR fusions with the inverse phenotype will allow the establishment of oriP-Mk ⁇ plasmids only in the presence of tetracycline or its derivatives, which is an even more stringent condition for plasmid gene vectors regulated in cis.
• the inventors developed a first generation of gene vector plasmids, which can be regulated in cis. These gene vector plasmids carry two marker genes for selection (puromycin or hygromycin resistance) and phenotypic tracing (GFP or mRFP) which can be easily replaced with genes of therapeutic interest.
• Such gene vector plasmids can be packaged into a viral particle when the necessary packaging signals are provided (Delecluse et al., 1999; Kreppel and Kochanek, 2004). Additional genes or even genetic loci of therapeutic interest can be added since the packaging capacity of DNA-based plasmid vectors or viral vectors can be large, exceeding 100 kbps (White et al., 2002).
• Somatic gene and immune therapy are in need of gene vector systems, which can be controlled by simple means and do not alter the recipient cell(s) genetically.
• the novel gene vector system presented herein offers both advantages and should contribute to the feasibility of innovative therapeutic approaches.
• the present system is providing the advantage of resulting in higher plasmid copy numbers as illustrated in Fig. 6.
• conditional gene vector system comprising the following elements:
• a vector carrying a cis-ac ⁇ ng element and one or more coding and/or non- coding sequences and b) a trans-acting factor having a DNA binding domain capable of binding to a region of the m-acting element and a domain capable of maintaining the protein in the nucleus of a target cell, characterized in that wherein the region of the cis-ac ⁇ ng element and the DNA binding domain of the trans ⁇ acting factor capable of binding to the region of the cis-ac ⁇ ng element are so selected that the nuclear maintenance of said vector carrying the cis-acX ⁇ ng element in a target cell may be regulated by adding an agent capable of interfering with the binding of said tr ⁇ ns-ac ⁇ ng factor to said m-acting element.
• maintenance refers to an important effect of this invention, i.e. the effect of keeping a vector in a desired target cell as long as this is wanted, for example, in an attempt of treating an animal or a human by gene therapy.
• Maintenance as used herein has a very specific meaning which should be clearly distinguished from related terms currently used in the field of science: by “maintenance” it is meant that an element, in the specific context of the present invention a /ra «s-acting factor, is firmly attached to the nucleus of a target cell, in particular to the chromatin scaffold of the nucleus.
• “Firmly attached” as used herein means that the respective element is kept in the cell during all stages of the cell cycle (Gl , S, and G2 stages of the cell cycle), in particular including mitosis.
• maintenance therefore, can also be defined as binding capability to mitotic chromatin. It is noted that the above term has to be strictly distinguished from other characteristics, which are, for example, shown by nuclear localization sequences (NLS). Many proteins, for example transcription factors and structural nuclear proteins, must move from the cytosol into the interior of the nucleus. They are targeted to the nucleus by their nuclear localization sequence. These proteins are actively transported through pores in the nuclear envelope into the interior. However, these NLS may only be regarded as being a transport signal and do not provide "maintenance” as explained herein, i.e. a firm (and reversible) attachment of the proteins to components of the nucleus of the target cell. Therefore, the effect of the vector system of the present invention can also be termed "plasmid maintenance and/or nuclear retention".
• c/s-acting element and "trans-acting factor” as used herein have the meaning as it is commonly used in the field of science.
• Os-acting elements are usually defined as DNA sequences in the vicinity of the structural portion of a gene or otherwise as a genetic unit that are functionally required. 7ra «s-acting factors are factors, usually considered to be proteins, that bind to the c/s-acting sequences to control gene expression, for example.
• EBNAl trans-ac ⁇ ng factor in the scope of definitions set forth herein is wt EBNAl mentioned above and discussed below, which is also illustrated in Fig. 2A.
• EBNAl is the wild-type EBNAl gene product of the prototype EBV strain B95.8 with designated functional domains and their amino acid residues shown.
• EBNAl has a DNA binding domain capable of binding to a region of the c/s-acting element, which, in this case, is the FR sequence of oriP.
• oriP is the corresponding cis-actmg element here.
• EBNAl furthermore, shows a domain capable of maintaining the protein in the nucleus of a target cell termed "chromatin association" in Fig. 2A.
• the EBV derived EBNAl-o ⁇ ' P system does not show the principle of the present invention, since the nuclear maintenance of a vector carrying the oriP cw-acting element in a target cell may not be regulated by adding an agent, in particular a small molecular weight compound, capable of interfering with the binding of EBNAl to oriP.
• an agent in particular a small molecular weight compound, capable of interfering with the binding of EBNAl to oriP.
• the or/P-EBNAl system does not work in the meaning of the present invention, however, may be modified as explained below in order to solve the problems posed herein.
• small molecular weight compounds as used herein is to be understood as a group of compounds having a molecular weight of between 50 and 2000, preferably 100-1000.
• the cis-actmg element used in the invention is derived from a naturally occurring czs-acting element, wherein the region provided for binding said m-acting element to the corresponding naturally occurring trans -acting factor is replaced by a heterologous DNA sequence, and that the tr ⁇ ns-ac ⁇ ng factor is derived from a naturally occurring tr ⁇ ns-actmg factor, in which the DNA binding domain capable of binding to a region of the cis-ac ⁇ ng element is replaced by a heterologous amino acid sequence.
• the above indicated modification of the OnP-EBNAl system may serve as an example. Since the binding between oriP and EBNAl may not be interfered or disrupted by an interfering agent, both sequences, i.e. the DNA binding domain capable of binding to a region of the FR region of oriP are replaced by a heterologous sequence, for example by TetR, and the FR region of oriP is replaced by a tetO fragment accordingly.
• a vector system will result having all elements as indicated in the first aspect of the invention, i.e. may be regulated in vivo and in vitro by a interfering agent, in this specific case by doxycycline.
• heterologous sequence is not specifically limited and comprises the replacement of the naturally occuring sequences by all other sequences, whether they were derived from a related or unrelated source (in the case of EBV, it may, e.g., be derived from another virus, or as explained above, from a procaryotic source).
• the c/s-acting element is naturally occurring and selected from the plasmid origin of DNA replication of Epstein-Barr virus, termed oriP, or from DNA motifs to which trans-acting factors bind site-specifically such as l ⁇ cO operator, GAL4 binding sites, OR1/2, tetO operator, or lexA operator, which can be found in E. coli, Saccharomyces ccrevisiae, phage lambda, transposon TnI O, and E. coli, respectively.
• the DNA binding domain of the tr ⁇ ns-acting factor is preferably selected from EBNAl, Lad repressor, GAL4 protein, Cro protein, the LexA protein or Tet repressor, which can be found in Epstein-Barr virus, E. coli, Saccharomyces cerevisiae, phage lambda, transposon TnIO, E. coli, or humans, respectively.
• the domain capable of maintaining the trans-acting factor in the nucleus of a target cell is preferably selected from EBNAl, Histone Hl or HMGAIa.
• the binding region of the c/s-acting element contains one or more tetO (tet operator), l ⁇ cO (lac operator) sites or other operator sites derived from m-acting elements not present in the target cell of the gene vector.
• This restriction must be considered in order to avoid an inappropriate site specific binding of the /raws-acting factor of the invention in the target cell (leading to a binding competition with the vector carrying the m-acting element of the invention).
• tetO turned out to be preferred in the present invention, since its binding to TetR may be disrupted in the presence of doxycycline as an interfering agent.
• the vector system of the invention preferably comprises a binding region of the cis- acting element which consists of at least 5 x tetO to 200 x tetO, preferably 10 x tetO to 100 x tetO, more preferably 20 to 40 x tetO sites or at least 5 x lacO to 200 x lacO, preferably 10 x lacO to 100 x lacO, preferably 20 to 40 x lacO sites.
• the DNA binding domain of the trans-acimg factor preferably is TetR (Tet Repressor) or Lad (Lac Repressor) if used in connection with the above referenced binding regions of the ex ⁇ acting elements.
• the trans ⁇ acting factor is derived from EBNAl , in which the carboxy-terminal sequences were replaced by TetR or Lad.
• the carboxy- terminal sequences may be defined as amino acids 379-641, more preferably 459-604 as depicted in Fig. 2 A.
• the /raws-acting factor preferably takes the form EBNAl :TetR (see Fig. 2B).
• the tr ⁇ ns-ac ⁇ ng factor is HMGAl or histone Hl fused to TetR or Lad, for example TetR:TetR:HMGAl a.
• the vector carrying the cis-ac ⁇ ng element and one or more coding and/or non- coding sequences, and used in the present vector system is a plasmid.
• the coding sequence carried in the vector is preferably selected from the group consisting of antigens, selectable and phenotypic marker proteins and genes complementing a somatic genetic defect in the target cell(s) of the gene vector.
• Somatic genetic defects in these genes (and diseases) and genes complementing same may be selected from the following: hemoglobin alpha, beta, gamma, delta (thalassemia); common gamma-chain cytokine receptor (SCID); adenosin deaminase (ADA immune deficiency); Bruton's tyrosine kinase (B-cell immune deficiency); clotting factor VIII (hemophilia A); clotting factor IX (hemophilia B) and lysosomal storage disorders (Morbus Gaucher, mucopolysaccharidosis, etc.).
• the vector may carry coding sequences of about 120 kb.
• the vector may additionally contain further non-coding sequences, for example promoter sequences in order to control transcription of the one or more coding sequences contained therein.
• All essential elements of the vector system of the invention are preferably present on one single vector.
• An exemplary and preferred vector of this kind is depicted in Fig. 7. It carries the cis-actmg replicon and the trans-acting factor TetR:TetR:HMGAla, which are indicated in the center of the map.
• Two additional marker genes coding for destabilized GFP and hygromycin B phosphotransferase are functional in metazoen cells whereas the gene ⁇ - lactamase confers ampicillin resistance in E. coli.
• vector system since it may provide the two essential elements, i.e. cis-actmg and trans acting element separately or combined in one single vector only.
• the interfering agent as used in the present invention is selected in order to be adapted to the precise cis-tr ⁇ ns-system used, but preferably, may be selected from doxycycline, tetracycline, isopropyl-beta-D-thiogalactopyranoside (IPTG), complexing metal ions, preferably Zn 2+ , or hormons.
• the invention provides a host cell, which has been transfected with a vector as defined hereinabove.
• the trans-acting factor is also encoded by this vector.
• the host cell preferably is a human or animal stem cell, preferably a hematopoietic stem cell, a T cell, or a B cell, or, for example, a human embryonic stem cell.
• the invention provides a combined preparation comprising: a) the vector system as defined above and b) the intervening agent as defined herein.
• components a) and b) are intended for subsequent administration (will be further explained below).
• the invention provides a pharmaceutical composition containing the vector system, a host cell or a combined preparation as disclosed herein, and a pharmaceutically acceptable carrier or diluent.
• ingredients of the present invention are preferably used in form of a pharmaceutical composition where they are mixed with suitable carriers or excipients in doses to treat or ameliorate the disease.
• a composition may also contain (in addition to the ingredient and the carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials well known in the art.
• pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).
• the characteristics of the carrier will depend on the route of administration.
• the pharmaceutical composition may further contain other agents which either enhance the activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect or to minimize side-effects.
• compositions are to be used for medical purposes, they will contain a therapeutically effective dose of the respective ingredient.
• a therapeutically effective dose further refers to that amount of the compound/ingredient sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of such conditions.
• Intravenous administration of the pharmaceutical composition of the invention to a patient is preferred.
• the invention is directed to the use of the vector system or a pharmaceutical composition for the manufacture of a medicine for the treatment of hemophilia, diabetes, rheumatoid arthritis, genetic immunodeficiency, and graft versus host disease.
• the invention further pertains to a method of treating a patient including the step of administering a therapeutically effective dose of a pharmaceutical composition as disclosed herein to a patient in need of said treatment.
• a treatment method follows the following protocol:
• a vector preferably comprising all essential and further non-essential elements of the invention placed on a single vector, is transfered into a host cell, preferably a hematopoietic stem cell.
• the host cells and vectors contained therein are propagated in vitro and subsequently, are admnistered to a patient suffering from a certain disease.
• the expressed trans -acting element via its DNA binding domain, binds to the binding region of the cis-ac ⁇ ng element on the vector, thereby attaching the vector to the nucleus of the target cell in the patient via its maintenance domain.
• the genetic information is neither rapidly lost through spontaneous degradation by cellular nucleases nor maintained by integrating the genetic information into the chromosome of the recipient cell. In contrast, it is reversibly maintained in the nucleus of the target cell.
• the coding sequence is expressed, therefore providing the therapeutically needed product to the patient. If the treatment is complete or if it is to be interrupted, the interfering agent is adminstered to the patient, leading to a disruption of the binding of the /raws-acting element to the m-acting element and, thus, releases the vector, which then is removed from the target cell by degradation of cellular nucleases.
• all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
• Fig. 1 Schematic overview of the oriP wild-type (wt) replicon and the hybrid replicon 20xtetO.
• the viral replicon oriP is shown with the two elements 'family of repeats' and 'dyad symmetry', which carry 20 and 4 EBNAl binding sites, respectively.
• Each EBNAl binding site is 30 bps in size; the so-called rep* element is auxiliary and can contribute to DNA replication of oriP plasmids under certain conditions.
• the modification of the hybrid replicon 20xtetO constitutes an array of twenty tetO binding sites (5'-TCCCTATCAGTGATAGAGA-S ') to which the TetR protein binds with high affinity.
• Fig. 2 Design of /r ⁇ «s-acting factors and cell lines.
• A Shown is the wild-type EBNAl gene product of the prototype EBV strain B95.8 with designated functional domains and their amino acid residues.
• B The fusion gene product EBNAl :TetR with the amino- terminal domain of EBNAl joined via an artificial linking domain [(SGa) 5 ] to the complete coding region of the tetR gene followed by the domain F, a transcriptional activation domain derived from VP 16 (Krueger et al., 2003).
• ⁇ a transcriptional activation domain
• Fig. 3 TetR DNA binding domain fused to EBNAl confers plasmid maintenance.
• the test plasmids 2832 (oriP wt) and 2835 (20xtetO) were separately transfected into the HEK. cell line expressing both EBNAl and EBNAl :TetR.
• the cells were cultivated in the presence of selective concentrations of puromycin (125ng/ml) with or without 2.0 ⁇ g/ml doxycycline (DOX) for a few days until the cells had reached confluence.
• puromycin 125ng/ml
• DOX 2.0 ⁇ g/ml doxycycline
• Low molecular weight DNA was isolated according to the Hirt procedure, cleaved with a restriction enzyme to linearize the test plasmids and Dpnl to digest unreplicated plasmids, which retain the dam methylation pattern that the plasmids had acquired during propagation in E. coli.
• Full-length .Dprcl-resistant plasmid DNA was detected by Southern blot analysis with a radioactive probe, which hybridizes to the prokaryotic plasmid backbone of the test plasmids 2832 and 2835, only.
• Fig. 4 Doxycycline interferes with short-term plasmid maintenance period.
• Three different HEK.293 cell lines, which express no viral protein control, top row panels), EBNAl (middle row panels), and both EBNAl plus EBNAl :TetR (bottom row panels) were used in this experiment.
• Two different test plasmids were examined which encode red fluorescence protein (mRFP).
• the plasmid 3154 carries wild-type oriP whereas 3155 is a test plasmid with the hybrid origin and 20 tetO sites (Tab. 1).
• the three cell lines were transfected with the test plasmids and selected with puromycin for two to four days, only, until most of the adherent cells appeared bright red under UV light in the microscope (day 0).
• the selective pressure was alleviated and the cells were kept in normal cell culture media with or without doxycycline (2.0 ⁇ g/ml) for a period of 12 days. No or only very few cells appeared to express mRFP in parental HEK293 cells transfected with the two test plasmids at that time point (top row and data not shown) indicating that both test plasmids were rapidly lost in the absence of EBNAl and EBNAl :TetR.
• HEK293 cells expressing EBNAl and transfected with the oriP wt test plasmid 3154 a considerable fraction of cells still expressed mRFP in the presence or absence of doxycycline (middle row, +/- DOX) after 12 days.
• HEK293 cells expressing both trans-factors and transfected with the hybrid replicon test plasmid 3155 were mRFP positive, only, when the cells had been cultivated in the absence of doxycycline (bottom row).
• Fig. 5 Hybrid origin plasmids are precipitously lost in the presence of doxycycline.
• the oriP wt (3230) plasmid and the two hybrid origin plasmids IQxtetO (3231) and (3293) were transfected into HEK293 cells expressing both EBNAl and TetR:TetR:HMGAl a (Fig. 2 E) and selected with hygromycin (80 ⁇ g/ml) for 19 days. At that time point (day 0) selection was removed and the cells were further cultivated with or without doxycycline (DOX, 2.5 ⁇ g/ml) for 12 days. Low molecular weight DNA was prepared according to the Hirt procedure at days 8 and 12.
• Hirt-extracted DNAs were also prepared from the same cells, which were kept under continuous selection with hygromycin.
• the DNAs were cleaved with Dpn ⁇ to digest traces of unreplicated plasmid DNAs and a restriction enzyme (PmIl) that cleaves cellular DNA, only, and analyzed after electrophoresis on an agarose gel by Southern blot hybridization with a radioactive probe, which hybridizes to the prokaryotic backbone of the gene vector plasmids.
• the control lane indicates the migration pattern of 500pg DNA of E.
• co//-derived control plasmid 3230 with the positions of the monomelic supercoiled, covalently closed, circular (ccc), monomelic open circles (oc), and multimers (mult.) forms of plasmid DNA.
• the ccc and oc DNA forms indicate the presence of extrachromosomal plasmids throughout the experiment.
• the hybrid origin plasmids 20xtetO and ⁇ QxtetO are precipitously lost on day 8 and 12 but are maintained at higher copy numbers than wt oriP with or without hygromycin selection in the absence of doxycycline.
• the oriP wt (3230) plasmid and the two hybrid origin plasmids 20xtetO (3231 ) and AQxtetO (3293) were transfected into HEK293 cells expressing both EBNAl and TetR:TetR:HMGAla and treated as described in Fig. 5. At days 4, 8, and 12 after omission of hygromycin from the cell culture media, the cells were analyzed by FACS for the expression of GFP encoded by all three plasmids.
• Hirt-extracted DNAs were prepared and cleaved with Dpnl as described in Fig. 5. 600ng DNA of each sample was transformed via electroporation into competent DHlOB E. coli cells. After phenotypic expression, different fractions of the E. coli cultures were plated on LB plates containing ampicillin (lOO ⁇ g/ml) and incubated at 30°C for 20 hours. Colonies were counted and the total number of ampicillin-resistant cells were calculated in these plasmid rescue experiments. The colony number are provided for each of the three test plasmids with or without doxycycline (DOX) as indicated.
• DOX doxycycline
• Fig. 7 Genetic map of the plasmid 3333. Shown is a gene vector plasmid 3333, which replicates and is maintained extrachromosomally in parental HEK293 cells. The ex ⁇ acting replicon and the trans- ⁇ c ⁇ mg factor TetR:TetR:HMGAl a are indicated in the center of the map. Two additional marker genes coding for destabilized GFP and hygromycin B phosphotransferase are functional in metazoen cells whereas the gene ⁇ - lactamase confers ampicillin resistance in E. coli.
• the prokaryotic TetR DNA binding domain fused to EBNAl confers plasmid maintenance.
• the DNA binding domain of EBNAl was deleted and replaced with the entire open reading frame of the prokaryotic DNA binding protein TetR. Similar to EBNAl , the tetR gene product forms homodimers and binds to its cognate DNA motif with very high affinity. In order to replace the acidic transactivation domain of EBNAl at its distal carboxy-terminal end the inventors used a functionally equivalent domain at the same location (domain F in Fig. 2B)(Krueger et al., 2003). This chimeric protein was called EBNAl :TetR.
• the EBNAl: tetR gene expressed from a heterologous promoter was stably integrated with conventional methods in the HEK293 human cell line that already expresses wild-type EBNAl as well. As shown in Fig. 2D, this cell line expresses both proteins.
• the inventors replaced the entire array of FR involved in plasmid maintenance with an identical number of direct repeats to which TetR binds with high affinity.
• This test plasmid called 2835 (Tab. 1) also carries a selectable maker gene for puromycin resistance and GFP as a phenotypic marker.
• the parental plasmid 2832 is identical to 2835 except that it carries wild-type oriP.
• test plasmids 2832 (oriP wt) and 2835 (20xtetO) were separately transfected into the HEK cell line expressing both EBNAl and EBNAl :TetR.
• the cells were cultivated in the presence of selective concentrations of puromycin (125ng/ml) with or without 2.0 ⁇ g/ml doxycycline (DOX) for a few days until the cells had reached confluence.
• Low molecular weight DNA was isolated according to the Hirt procedure, cleaved with a restriction enzyme to linearize the test plasmids and Dpn ⁇ to digest unreplicated plasmids, which retain the dam methylation pattern that the plasmids had acquired during propagation in E. coli.
• test plasmid 2835 which carries a hybrid replicon with a multimer of 20 tetO sites at the position of the FR array can replicate and is maintained and (ii) doxycycline, which interferes with DNA binding of TetR abrogates the maintenance of this plasmid vector.
• the inventors used three different cell lines, which express no viral protein (parental HEK293 cells), EBNAl, and both EBNAl plus EBNAl :TetR.
• three different test plasmids were used which both encode red fluorescence protein (mRFP)(Campbell et al., 2002).
• the plasmid 3154 carries wild-type oriP whereas 3 155 is a test plasmid with the hybrid origin and 20 tetO sites (Tab. 1).
• the three cell lines were transfected with the test plasmids and selected with puromycin for two to four days, only, until most of the adherent cells appeared bright red under UV light in the microscope (day 0, Fig. 4).
• the selective pressure was alleviated and the cells were kept in normal cell culture media with or without doxycycline (2.0 ⁇ g/ml) for a period of 12 days. No or only very few cells appeared to express mRFP in parental HEK293 cells transfected with the two test plasmids at that time point (Fig. 4 top row and data not shown) indicating that both test plasmids were rapidly lost in the absence of EBNAl and EBNAl :TetR.
• HEK293 cells expressing EBNAl and transfected with the oriP wt test plasmid 3154 a considerable fraction of cells still expressed mRFP in the presence or absence of doxycycline (+/- DOX in Fig. 4, middle row) after 12 days.
• HEK293 cells expressing both /r ⁇ «5-factors and transfected with the hybrid replicon test plasmid 3155 were mRFP positive, only, when the cells had been cultivated in the absence of doxycycline (Fig. 4, bottom row).
• the amino-terminal part in the EBNAl :TetR fusion protein is expected to associate to chromatin, which is presumably essential for plasmid maintenance. Since wild-type oriP plasmids replicate and are maintained stably in the presence of another chimeric protein, HMGAl a:EBNAl (Hung et al., 2001), we argued that a novel chimeric protein consisting of the coding sequence of HMGAIa fused to the TetR DNA-binding gene might be functional as well.
• An expression cassette with the HMGAl a:TetR gene was stably integrated into the parental cell line HEK293 EBNAl (Fig. 2E).
• plasmid DNAs could be detected by Southern blot hybridization (Fig. 5) and plasmid rescue in E. coli (Fig. 6). The majority of the cell pools expressed GFP as expected (Fig. 6). It was immediately apparent, that both hybrid plasmids (20xtetO and ⁇ OxtetO) were maintained as extrachromosomal DNAs at higher copy numbers than oriP wt (Fig. 5 and 6), although the cells did not express higher amounts of GFP (Fig. 6). Upon removal of hygromycin selection, the GFP expression levels gradually diminished in all cell pools (Fig. 6).
• Table. 1 Overview of different gene vector plasmids
• the plasmid replicon of EBV consists of multiple c/s-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element.
• Hacein-Bey-Abina S., Von Kalle, C, Schmidt, M., McCormack, M.P., Wulffraat, N., Leboulch, P., Lim, A., Osborne, C. S., Pawliuk, R., Morillon, E., Sorensen, R., Forster, A., Fraser, P., Cohen, J.

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